Method and apparatus for imaging a sample on a device

ABSTRACT

Labeled targets on a support synthesized with polymer sequences at known locations according to the methods disclosed in U.S. Pat. No. 5,143,854 and PCT WO 92/10092 or others, can be detected by exposing selected regions of sample 1500 to radiation from a source 1100 and detecting the emission therefrom, and repeating the steps of exposition and detection until the sample is completely examined.

This is a Division of application Ser. No. 08/708,335 filed Sep. 4,1996, now U.S. Pat. No. 5,834,758 which is a continuation of Ser. No.08/301,051, filed Sep. 2, 1994 and is now U.S. Pat. No. 5,578,832.

REFERENCE TO A "MICROFICHE APPENDIX"

This application contains a microfiche appendix. The total number ofmicrofiche is 2 and the total number of frames is 82.

COPYRIGHT NOTICE

A portion of the disclosure of this patent document contains materialwhich is subject to copyright protection. The copyright owner has noobjection to the facsimile reproduction by anyone of the patent documentor the patent disclosure as it appears in the Patent and TrademarkOffice patent file or records, but otherwise reserves all copyrightrights whatsoever.

BACKGROUND OF THE INVENTION

The present invention relates to the field of imaging. In particular,the present invention provides methods and apparatus for high speedimaging of a sample containing labeled markers with high sensitivity andresolution.

Methods and systems for imaging samples containing labeled markers suchas confocal microscopes are commercially available. These systems,although capable of achieving high resolution with good depthdiscrimination, have a relatively small field of view. In fact, thesystem's field of view is inversely related to its resolution. Forexample, a typical 40× microscope objective, which has a 0.25 μmresolution, has a field size of only about 500 μm. Thus, confocalmicroscopes are inadequate for applications requiring high resolutionand large field of view simultaneously.

Other systems, such as those discussed in U.S. Pat No. 5,143,854(Pirrung et al.), PCT WO 92/10092, and U.S. Pat. No. 5,631,734,incorporated herein by reference for all purposes, are also known. Thesesystems include an optical train which directs a monochromatic orpolychromatic light source to about a 5 micron (μm) diameter spot at itsfocal plane. A photon counter detects the emission from the device inresponse to the light. The data collected by the photon counterrepresents one pixel or data point of the image. Thereafter, the lightscans another pixel as the translation stage moves the device to asubsequent position.

As disclosed, these systems resolve the problem encountered by confocalmicroscopes. Specifically, high resolution and a large field of view aresimultaneously obtained by using the appropriate objective lens andscanning the sample one pixel at a time. However, this is achieved bysacrificing system throughput. As an example, an array of materialformed using the pioneering fabrication techniques, such as thosedisclosed in U.S. Pat No. 5,143,854 (Pirrung et al.), U.S. patentapplication Ser. No. 08/143,312, now abandoned and U.S. patentapplication Ser. No. 08/255,682, now abandoned, incorporated herein byreference for all purposes, may have about 10⁵ sequences in an area ofabout 13 mm×13 mm. Assuming that 16 pixels are required for each memberof the array (1.6×10⁶ total pixels), the image can take over an hour toacquire.

In some applications, a full spectrally resolved image of the sample maybe desirable. The ability to retain the spectral information permits theuse of multi-labeling schemes, thereby enhancing the level ofinformation obtained. For example, the microenvironment of the samplemay be examined using special labels whose spectral properties aresensitive to some physical property of interest. In this manner, pH,dielectric constant, physical orientation, and translational and/orrotational mobility may be determined.

From the above, it is apparent that improved methods and systems forimaging a sample are desired.

SUMMARY OF THE INVENTION

Methods and systems for detecting a labeled marker on a sample locatedon a support are disclosed. The imaging system comprises a body forimmobilizing the support. Excitation radiation, from an excitationsource having a first wavelength, passes through excitation optics. Theexcitation optics cause the excitation radiation to excite a region onthe sample. In response, labeled material on the sample emits radiationwhich has a wavelength that is different from the excitation wavelength.Collection optics then collect the emission from the sample and image itonto a detector. The detector generates a signal proportional to theamount of radiation sensed thereon. The signal represents an imageassociated with the plurality of regions from which the emissionoriginated. A translator is employed to allow a subsequent plurality ofregions on said sample to be excited. A processor processes and storesthe signal so as to generate a 2-dimensional image of said sample.

In one embodiment, excitation optics focus excitation light to a line ata sample, simultaneously scanning or imaging a strip of the sample.Surface bound labeled targets from the sample fluoresce in response tothe light. Collection optics image the emission onto a linear array oflight detectors. By employing confocal techniques, substantially onlyemission from the light's focal plane is imaged. Once a strip has beenscanned, the data representing the 1-dimensional image are stored in thememory of a computer. According to one embodiment, a multi-axistranslation stage moves the device at a constant velocity tocontinuously integrate and process data. As a result, a 2-dimensionalimage of the sample is obtained.

In another embodiment, collection optics direct the emission to aspectrograph which images an emission spectrum onto a 2-dimensionalarray of light detectors. By using a spectrograph, a full spectrallyresolved image of the sample is obtained.

The systems may include auto-focusing feature to maintain the sample inthe focal plane of the excitation light throughout the scanning process.Further, a temperature controller may be employed to maintain the sampleat a specific temperature while it is being scanned. The multi-axistranslation stage, temperature controller, auto-focusing feature, andelectronics associated with imaging and data collection are managed byan appropriately programmed digital computer.

In connection with another aspect of the invention, methods foranalyzing a full spectrally resolved image are disclosed. In particular,the methods include, for example, a procedure for deconvoluting thespectral overlap among the various types of labels detected. Thus, a setof images, each representing the surface densities of a particular labelcan be generated.

A further understanding of the nature and advantages of the inventionsherein may be realized by reference to the remaining portions of thespecification and the attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a block diagram of an imaging system;

FIG. 2 illustrates how the imaging system achieves good depthdiscrimination;

FIG. 3 shows the imaging system according to the present invention;

FIGS. 4a-4d show a flow cell on which a substrate is mounted;

FIG. 5 shows a agitation system;

FIG. 6 is a flow chart illustrating the general operation of the imagingsystem;

FIGS. 7a-7b are flow charts illustrating the steps for focusing thelight at the sample;

FIG. 8 is a flow chart illustrating in greater detail the steps foracquiring data;

FIG. 9 shows an alternative embodiment of the imaging system;

FIG. 10 shows the axial response of the imaging system of FIG. 9;

FIGS. 11a-11b are flow charts illustrating the general operations of theimaging system according to FIG. 9;

FIGS. 12a-12b are flow charts illustrating the steps for plotting theemission spectra of the acquired image;

FIG. 12c shows the data structure of the data file according to theimaging system in FIG. 9;

FIG. 13 shows the normalized emission spectra of four fluorophores whoseemission spectra are shown in FIG. 17, and scaled according to the stepsset forth in FIG. 12a after they have been normalized;

FIG. 14 is a flow chart illustrating the steps for image deconvolution;

FIG. 15 shows the layout of the probe sample;

FIG. 16 shows examples of monochromatic images obtained by the imagingsystem of FIG. 9;

FIGS. 17-18 show the emission spectra obtained by the imaging system ofFIG. 9;

FIG. 19 shows the emission cross section matrix elements obtained fromthe emission spectra of FIG. 13;

FIG. 20 shows examples of images representing the surface density of thefluorophores; and

FIG. 21 shows an alternative embodiment of an imaging system.

DESCRIPTION OF THE PREFERRED EMBODIMENT Contents

I. Definitions

II. General

a. Introduction

b. Overview of the Imaging System

III. Detailed Description of One Embodiment of the Imaging System

a. Detection Device

b. Data acquisition

IV. Detailed Description of an Alternative Embodiment of the ImagingSystem

a. Detection Device

b. Data Acquisition

c. Postprocessing of the Monochromatic Image Set

d. Example of spectral deconvolution of a 4-fluorophore system

V. Detailed Description of Another Embodiment of the Imaging System

I. Definitions

The following terms are intended to have the following general meaningsas they are used herein:

1. Complementary: Refers to the topological compatibility or matchingtogether of interacting surfaces of a probe molecule and its target.Thus, the target and its probe can be described as complementary, andfurthermore, the contact surface characteristics are complementary toeach other.

2. Probe: A probe is a surface-immobilized molecule that is recognizedby a particular target. Examples of probes that can be investigated bythis invention include, but are not restricted to, agonists andantagonists for cell membrane receptors, toxins and venoms, viralepitopes, hormones (e.g., opioid peptides, steroids, etc.), hormonereceptors, peptides, enzymes, enzyme substrates, cofactors, drugs,lectins, sugars, oligonucleotides, nucleic acids, oligosaccharides,proteins, and monoclonal antibodies.

3. Target: A molecule that has an affinity for a given probe. Targetsmay be naturally-occurring or manmade molecules. Also, they can beemployed in their unaltered state or as aggregates with other species.Targets may be attached, covalently or noncovalently, to a bindingmember, either directly or via a specific binding substance. Examples oftargets which can be employed by this invention include, but are notrestricted to, antibodies, cell membrane receptors, monoclonalantibodies and antisera reactive with specific antigenic determinants(such as on viruses, cells or other materials), drugs, oligonucleotides,nucleic acids, peptides, cofactors, lectins, sugars, polysaccharides,cells, cellular membranes, and organelles. Targets are sometimesreferred to in the art as anti-probes. As the term targets is usedherein, no difference in meaning is intended. A "Probe Target Pair" isformed when two macromolecules have combined through molecularrecognition to form a complex.

II. General

a. Introduction

The present invention provides methods and apparatus for obtaining ahighly sensitive and resolved image at a high speed. The invention willhave a wide range of uses, particularly, those requiring quantitativestudy of a microscopic region from within a larger region, such as 1 μm²over 100 mm². For example, the invention will find application in thefield of histology (for studying histochemical stained and immunologicalfluorescent stained images), video microscopy, or fluorescence in situhybridization. In one application, the invention herein is used to imagean array of probe sequences fabricated on a support.

The support on which the sequences are formed may be composed from awide range of material, either biological, nonbiological, organic,inorganic, or a combination of any of these, existing as particles,strands, precipitates, gels, sheets, tubing, spheres, containers,capillaries, pads, slices, films, plates, slides, etc. The substrate mayhave any convenient shape, such as a disc, square, sphere, circle, etc.The substrate is preferably flat but may take on a variety ofalternative surface configurations. For example, the substrate maycontain raised or depressed regions on which a sample is located. Thesubstrate and its surface preferably form a rigid support on which thesample can be formed. The substrate and its surface are also chosen toprovide appropriate light-absorbing characteristics. For instance, thesubstrate may be a polymerized Langmuir Blodgett film, functionalizedglass, Si, Ge, GaAs, GaP, SiO₂, SiN₄, modified silicon, or any one of awide variety of gels or polymers such as (poly)tetrafluoroethylene,(poly)vinylidenedifluoride, polystyrene, polycarbonate, or combinationsthereof. Other substrate materials will be readily apparent to those ofskill in the art upon review of this disclosure. In a preferredembodiment the substrate is flat glass or silica.

According to some embodiments, the surface of the substrate is etchedusing well known techniques to provide for desired surface features. Forexample, by way of the formation of trenches, v-grooves, mesastructures, or the like, the synthesis regions may be more closelyplaced within the focus point of impinging light. The surface may alsobe provided with reflective "mirror" structures for maximization ofemission collected therefrom.

Surfaces on the solid substrate will usually, though not always, becomposed of the same material as the substrate. Thus, the surface may becomposed of any of a wide variety of materials, for example, polymers,plastics, resins, polysaccharides, silica or silica-based materials,carbon, metals, inorganic glasses, membranes, or any of the above-listedsubstrate materials. In one embodiment, the surface will be opticallytransparent and will have surface Si-OH functionalities, such as thosefound on silica surfaces.

The array of probe sequences may be fabricated on the support accordingto the pioneering techniques disclosed in U.S. Pat. No. 5,143,854, PCTWO 92/10092, or U.S. application Ser. No. 07/624,120, filed Dec. 6, 1990(now abandoned), incorporated herein by reference for all purposes. Thecombination of photolithographic and fabrication techniques may, forexample, enable each probe sequence ("feature") to occupy a very smallarea ("site") on the support. In some embodiments, this feature site maybe as small as a few microns or even a single molecule. For example,about 10⁵ to 10⁶ features may be fabricated in an area of only 12.8 mm².Such probe arrays may be of the type known as Very Large ScaleImmobilized Polymer Synthesis (VLSIPS™).

The probe arrays will have a wide range of applications. For example,the probe arrays may be designed specifically to detect geneticdiseases, either from acquired or inherited mutations in an individualDNA. These include genetic diseases such as cystic fibrosis, diabetes,and muscular dystrophy, as well as acquired diseases such as cancer(P53-gene relevant to some cancers), as disclosed in U.S. patentapplication Ser. No. 08/143,312, already incorporated by reference.

Genetic mutations may be detected by a method known as sequencing byhybridization. In sequencing by hybridization, a solution containing oneor more targets to be sequenced (i.e., samples from patients) contactsthe probe array. The targets will bind or hybridize with complementaryprobe sequences. Generally, the targets are labeled with a fluorescentmarker, radioactive isotopes, enzymes, or other types of markers.Accordingly, locations at which targets hybridize with complimentaryprobes can be identified by locating the markers. Based on the locationswhere hybridization occur, information regarding the target sequencescan be extracted. The existence of a mutation may be determined bycomparing the target sequence with the wild type.

The interaction between targets and probes can be characterized in termsof kinetics and thermodynamics. As such, it may be necessary tointerrogate the array while in contact with a solution of labeledtargets. Consequently, the detection system must be extremely selective,with the capacity to discriminate between surface-bound andsolution-born targets. Also, in order to perform a quantitativeanalysis, the high-density volume of the probe sequences requires thesystem to have the capacity to distinguish between each feature site.

b. Overview of the Imaging System

An image is obtained by detecting the electromagnetic radiation emittedby the labels on the sample when it is illuminated. Emission fromsurface-bound and solution-free targets is distinguished through theemployment of confocal and auto-focusing techniques, enabling the systemto image substantially only emission originating from the surface of thesample. Generally, the excitation radiation and response emission havedifferent wavelengths. Filters having high transmissibility in thelabel's emission band and low transmissibility in the excitationwavelength may be utilized to virtually eliminate the detection ofundesirable emission. These generally include emission from out-of-focusplanes or scattered excitation illumination as potential sources ofbackground noise.

FIG. 1 is an optical and electronic block diagram illustrating theimaging system according to the present invention. Illumination of asample 1500 may be achieved by exposing the sample to electromagneticradiation from an excitation source 1100. Various excitation sources maybe used, including those which are well known in the art such as anargon laser, diode laser, helium-neon laser, dye laser, titaniumsapphire laser, Nd:YAG laser, arc lamp, light emitting diodes, anyincandescent light source, or other illuminating device.

Typically, the source illuminates the sample with an excitationwavelength that is within the visible spectrum, but other wavelengths(i.e., near ultraviolet or near infrared spectrum) may be used dependingon the application (i.e., type of markers and/or sample). In someembodiments, the sample is excited with electromagnetic radiation havinga wavelength at or near the absorption maximum of the species of labelused. Exciting the label at such a wavelength produces the maximumnumber of photons emitted. For example, if fluorescein (absorptionmaximum of 488 nm) is used as a label, an excitation radiation having awavelength of about 488 nm would induce the strongest emission from thelabels.

In instances where a multi-labeling scheme is utilized, a wavelengthwhich approximates the mean of the various candidate labels' absorptionmaxima may be used. Alternatively, multiple excitations may beperformed, each using a wavelength corresponding to the absorptionmaximum of a specific label. Table I lists examples of various types offluorophores and their corresponding absorption maxima.

                  TABLE I                                                         ______________________________________                                        Candidate Fluorophores                                                                         Absorption Maxima                                            ______________________________________                                        Fluorescein      488 nm                                                         Dichloro-fluorescein 525 nm                                                   Hexachloro-fluorescein 529 nm                                                 Tetramethylrhodamine 550 nm                                                   Rodamine X 575 nm                                                             Cy3 ™ 550 nm                                                               Cy5 ™ 650 nm                                                               Cy7 ™ 750 nm                                                               IRD40 785 nm                                                                ______________________________________                                    

The excitation source directs the light through excitation optics 1200,which focus the light at the sample. The excitation optics transform thelight into a "line" sufficient to illuminate a row of the sample.Although the Figure illustrates a system that images one vertical row ofthe sample at a time, it can easily be configured to image the samplehorizontally or to employ other detection scheme. In this manner, a rowof the sample (i.e., multiple pixels) may be imaged simultaneously,increasing the throughput of the imaging systems dramatically.

Generally, the excitation source generates a beam with a Gaussianprofile. In other words, the excitation energy of the line peaks flatlynear the center and diminishes therefrom (i.e., non-uniform energyprofile). Illuminating the sample with a non-uniform energy profile willproduce undesirable results. For example, the edge of the sample that isilluminated by less energetic radiation would appear more dim relativeto the center. This problem is resolved by expanding the line to permitthe central portion of the Gaussian profile to illuminate the sample.

The width of the line (or the slit aperture) determines the spatialresolution of the image. The narrower the line, the more resolved theimage. Typically, the line width is dictated by the feature size ofsample. For example, if each probe sequence occupies a region of about50 μm, then the minimum width is about 50 μm. Preferably, the widthshould be several times less than the feature size to allow foroversampling.

Excitation optics may comprise various optical elements to achieved thedesired excitation geometry, including but not limited to microscopeobjectives, optical telescopes, cylindrical lens, cylindricaltelescopes, line generator lenses, anamorphic prisms, combination oflenses, and/or optical masks. The excitation optics may be configured toilluminate the sample at an angle so as to decouple the excitation andcollection paths. As a result, the burden of separating the light pathsfrom each other with expensive dichroic mirrors or other filters isessentially eliminated. In one embodiment, the excitation radiationilluminates the sample at an incidence of about 45°. This configurationsubstantially improves the system's depth discrimination since emissionfrom out-of-focus planes is virtually undetected. This point willsubsequently be discussed in more detail in connection with FIG. 2.

As the incident light is reflected from the sample, it passes throughfocusing optics 1400, which focus the reflected illumination line to apoint. A vertical spatial slit 1405 and light detector 1410 are locatedbehind the focusing optics. Various light detectors may be used,including photodiodes, avalanche photodiodes, phototransistors, vacuumphotodiodes, photomultiplier tubes, and other light detectors. Thefocusing optics, spatial slit, and light detector serve to focus thesample in the focal plane of the excitation light. In one embodiment,the light is focused at about the center of the slit when the sample islocated in the focal plane of the incident light. Using the lightdetector to sense the energy, the system can determine when the sampleis in focus. In some applications, the slit may be eliminated byemploying a split photodiode (bi-cell or quadrant detector),position-sensitive photodiode, or position-sensitive photomultiplier.

The line illumination technique presents certain concerns such asmaintaining the plane of the sample perpendicular to the optical axis ofthe collection optics. If the sample is not aligned properly, imagedistortion and intensity variation may occur. Various methods, includingshims, tilt stage, gimbal mount, goniometer, air pressure or pneumaticbearings or other technique may be employed to maintain the sample inthe correct orientation. In one embodiment, a beam splitter 1420 may bestrategically located to direct a portion of the beam reflected from thesample. A horizontal spatial slit 1425 and light detector 1430, similarto those employed in the auto-focusing technique, may be used to sensewhen the plane of the sample is perpendicular to the optical axis of thecollection optics.

In response to the excitation light, the labeled targets fluoresce(i.e., secondary radiation). The emission, is collected by collectionoptics 1300 and imaged onto detector 1800. A host of lenses orcombination of lenses may be used to comprise collection optics, such ascamera lenses, microscope objectives, or a combination of lenses. Thedetector may be an array of light detectors used for imaging, such ascharge-coupled devices (CCD) or charge-injection devices (CID). Otherapplicable detectors may include image-intensifier tubes, image orthicontube, vidicon camera type, image dissector tube, or other imagingdevices. Generally, the length of the CCD array is chosen tosufficiently detect the image produced by the collection optics. Themagnification power of the collection optics dictates the dimension ofthe image. For instance, a 2× collection optics produces an image equalto about twice the height of the sample.

The magnification of the collection optics and the sensitivity ofdetector 1800 play an important role in determining the spatialresolution capabilities of the system. Generally, the spatial resolutionof the image is restricted by the pixel size of detector 1800. Forexample, if the size of each pixel in the detector is 25 μm², then thebest image resolution at 1× magnification is about 25 μm. However, byincreasing the magnification power of the collection optics, a higherspatial resolution may be achieved with a concomitant reduction of fieldof view. As an illustration, increasing the magnification of thecollection optics to 5 would increase the resolution by a factor of 5(from 25 μm to 5 μm).

A filter, such as a long pass glass filter, long pass or band passdielectric filter, may be located in front of detector 1800 to preventimaging of unwanted emission, such as incident light scattered by thesubstrate. Preferably, the filter transmits emission having a wavelengthat or greater than the fluorescence and blocks emission having shorterwavelengths (i.e., blocking emission at or near the excitationwavelength).

Once a row of fluorescent data has been collected or integrated), thesystem begins to image a subsequent row. This may be achieved bymounting the sample on a translation stage and moving it across theexcitation light. Alternatively, Galvo scanners or rotating polyhedralmirrors may be employed to scan the excitation light across the sample.A complete 2-dimensional image of the sample is generated by combiningthe rows together.

The amount of time required to obtain the 2-dimensional image depends onseveral factors, such as the intensity of the laser, the type of labelsused, the detector sensitivity, noise level, and resolution desired. Inone embodiment, a typical integration period of a single row may beabout 40 msec. Given that, a 14 μm resolution image of a 12.8 mm² samplecan be acquired in less than 40 seconds.

Thus, the present invention acquires images as fast as conventionalconfocal microscope while achieving the same resolution, but with a muchlarger field of view. In one dimension, the field of view is dictated bythe translation stage and can be arbitrarily large (determined by thedistance it translates during one integration period). In the otherdimension, the field of view is limited by the objective lens. However,this limitation may be eliminated employing a translation stage for thatdimension.

FIG. 2 is a simplified illustration exhibiting how the imaging systemachieves good depth discrimination. As shown, a focal plane 200 islocated between planes 210 and 220. Planes 210 and 220 both representplanes that are out of focus. In response to the incident light 250, all3 planes fluoresce light. This emission is transmitted throughcollection optics 261. However, emission originating from out-of-focusplanes 210 and 220 is displaced sideways at 211 and 221, respectively,in relationship to the collection optics' optical axis 280. Since theactive area of the light detectors array 260 is about 14 μm wide, nearlyall of the emission from any plane that is more than slightlyout-of-focus is not detected.

III. Detailed Description of One Embodiment of the Imaging System.

a. Detection Device

FIG. 3 schematically illustrates a particular system for imaging asample. The system includes a body 3220 for holding a support 130containing the sample on a surface 131. In some embodiments, the supportmay be a microscope slide or any surface which is adequate to hold thesample. The body 3220, depending on the application, may be a flow cellhaving a cavity 3235. Flow cells, such as those disclosed in U.S. patentapplication Ser. No. 08/255,682, already incorporated by reference, mayalso be used. The flow cell, for example, may be employed to detectreactions between targets and probes. In some embodiments, the bottom ofthe cavity may comprise a light absorptive material so as to minimizethe scattering of incident light.

In embodiments utilizing the flow cell, surface 131 is mated to body3220 and serves to seal cavity 3235. The flow cell and the substrate maybe mated for sealing with one or more gaskets. In one embodiment, thesubstrate is mated to the body by vacuum pressure generated by a pump3520. Optionally, the flow cell is provided with two concentric gasketsand the intervening space is held at a vacuum to ensure mating of thesubstrate to the gaskets. Alternatively, the substrate may be attachedby using screws, clips, or other mounting techniques.

When mated to the flow cell, the cavity encompasses the sample. Thecavity includes an inlet port 3210 and an outlet port 3211. A fluid,which in some embodiments contains fluorescently labeled targets, isintroduced into the cavity through inlet port 3210. A pump 3530, whichmay be a model no. B-120-S made by Eldex Laboratories, circulates fluidsinto the cavity via inlet 3210 port and out through outlet port 3211 forrecirculation or disposal. Alternatively, a syringe, gas pressure, orother fluid transfer device may be used to flow fluids into and throughthe cavity.

Optionally, pump 3530 may be replaced by an agitation system thatagitates and circulates fluids through the cavity. Agitating the fluidsshortens the incubation period between the probes and targets. This canbe best explained in terms of kinetics. A thin layer, known as thedepletion layer, is located above the probe sample. Since targetsmigrate to the surface and bind with the probe sequences, this layer isessentially devoid of targets. However, additional targets are inhibitedfrom flowing into the depletion layer due to finite diffusioncoefficients. As a result, incubation period is significantly increased.By using the agitation system to dissolve the depletion layer,additional targets are presented at the surface for binding. Ultrasonicradiation and/or heat, shaking the holder, magnetic beads, or otheragitating technique may also be employed.

In some embodiments, the flow cell is provided with a temperaturecontroller 3500 for maintaining the flow cell at a desired temperature.Since probe/target interaction is sensitive to temperature, the abilityto control it within the flow cell permits hybridization to be conductedunder optimal temperature. Temperature controller 3500, which is a model13270-615 refrigerated circulating bath with a RS232 interface made byVWR Scientific, controls temperature by circulating water at a specifiedtemperature through channels formed in the flow cell. A computer 3400,which may be any appropriately programmed digital computer, such as aGateway 486DX operating at 33 MHz, monitors and controls therefrigerated bath via the RS232 interface. Alternatively, a refrigeratedair circulating device, resistance heater, peltier device(thermoelectric cooler), or other temperature controller may beimplemented.

According to one embodiment, flow cell 3220 is mounted to a x-y-ztranslation stage 3245. Translation stage 3245, for example, may be aPacific Precision Laboratories Model ST-SL06R-B5M driven by steppingmotors. The flow cell may be mated to the translation stage by vacuumpressure generated by pump 3520. Alternatively, screws, clips or othermounting techniques may be employed to mate the flow cell to thetranslation stage.

As previously mentioned, the flow cell is oriented to maintain thesubstrate perpendicular to the optical axis of the collection optics,which in some embodiments is substantially vertical. Maintaining thesupport in the plane of the incident light minimizes or eliminates imagedistortion and intensity variations which would otherwise occur. In someembodiments, the x-y-z translation stage may be mounted on a tilt stage3240 to achieve the desired flow cell orientation. Alternatively, shimsmay be inserted to align the flow cell in a substantially verticalposition. Movement of the translation stage and tilt stage may becontrolled by computer 3400.

To initiate the imaging process, incident light from a light source 3100passes through excitation optics, which in turn focus the light at thesupport. In one embodiment, the light source is a model 2017 argon lasermanufactured by Spectra-Physics. The laser generates a beam having awavelength of about 488 nm and a diameter of about 1.4 mm at the 1/e²points. As the radial beam passes through the optical train, it istransformed into a line, for example, of about 50 mm×11 μm at the 1/e²points. This line is more than sufficient to illuminate the sample,which in some embodiments is about 12.8 mm, with uniform intensitywithin about 10%. Thus, potential image distortions or intensityvariations are minimized.

The various elements of the excitation optics underlying thetransformation of the beam into the desired spatial excitation geometrywill now be described. Light source 3100 directs the beam through, forexample, a 3× telescope 3105 that expands and collimates the beam toabout 4.2 mm in diameter. In some embodiments, the 3× telescope includeslenses 3110 and 3120, which may be a -25 mm focal length plano-concavelens and a 75 mm focal length plano-convex lens, respectively.Alternatively, the 3× telescope may comprise any combination of lenseshaving a focal length ratio of 1:3.

Thereafter, the beam passes through a cylindrical telescope 3135. Thecylindrical telescope, for example, may have a magnification power of12. In some embodiments, telescope 135 comprises a -12.7 mm focal lengthcylindrical lens 3130 and a 150 mm focal length cylindrical lens 3140.Alternatively, cylindrical telescope 3135 includes any combination ofcylindrical lenses having a focal length ratio of 1:12 or a 12×anamorphic prism pair. Cylindrical telescope 3135 expands the beamvertically to about 50 mm.

In another alternative, lens 3130 of telescope 3135 may be aline-generator lens, such as an acylindrical lens with one piano surfaceand a hyperbolic surface. The line-generator lens converts a gaussianbeam to one having uniform intensity along its length. When using aline-generator lens, the beam may be expanded to the height of thesample, which is about 13.0 mm.

Next, the light is focused onto the sample by a lens 3170. In someembodiments, lens 3170 may be a 75 mm focal length cylindrical lens thatfocuses the beam to a line of about 50 mm×11 μm at its focal plane.Preferably, the sample is illuminated at an external incident angle ofabout 45°, although other angles may be acceptable. As illustrated inFIG. 2, illuminating the sample at an angle: 1) improves the depthdiscrimination of the detection system; and 2) decouples theillumination and collection light paths. Optionally, a mirror 3160 isplaced between lens 3140 and lens 3170 to steer the beam appropriately.Alternatively, the mirror or mirrors may be optionally placed at otherlocations to provide a more compact system.

As depicted in the Figure, the incident light is reflected by thesubstrate through lenses 3350 and 3360. Lenses 3350 and 3360, forexample, may be a 75 mm focal length cylindrical lens and a 75 mm focallength spherical lens, respectively. Lens 3350 collimates the reflectedlight and lens 3360 focuses the collimated beam to about an 11 μm spotthrough a slit 3375. In some embodiments, the vertical slit may have awidth of about 25 μm. As the translation stage moves the substratethrough focus, the spot moves horizontally across the vertical slit. Inone embodiment, the optics are aligned to locate the spot substantiallyat the center of the slit when the substrate is located in the focalplane of the incident light.

A photodiode 3380 is located behind slit 3375 to detect an amount oflight passing through the slit. The photodiode, which may be a 13 DSI007made by Melles Griot, generates a voltage proportional to the amount ofthe detected light. The output from the photodiode aids computer 3400 infocusing the incident light at the substrate.

For embodiments employing a tilt stage 3240, a beam splitter 3390,horizontal slit 3365, and photodiode 3370 may be optionally configuredto detect when the substrate is substantially parallel to the plane ofthe incident light. Beam splitter 3390, which in some embodiments is a50% plate beam splitter, directs a portion of the reflected light fromthe substrate toward horizontal slit 3365. The horizontal slit may havea width of about 25 μm wide. As the tilt stage rotates the substratefrom the vertical plane, the beam spot moves vertically across thehorizontal slit. The beam splitter locates the spot substantially at thecenter of slit 3365 when the sample is substantially vertical.

A photodiode 3370, which may be similar to photodiode 3380, is locatedbehind slit 3375. The output from the photodiode aids computer 3400 inpositioning the substrate vertically.

In response to the illumination, the surface bound targets, which, forexample, may be labeled with fluorescein, fluoresce light. Thefluorescence is transmitted through a set of collection optics 3255. Insome embodiments, collection optics may comprise lenses 3250 and 3260,which may be 83 mm focal lengths f/1.76 lenses manufactured byRodenstock Precision Optics. In some embodiments, collection optics areconfigured at 1× magnification. In alternative embodiments, collectionoptics may comprise a pair of 50 mm focal length f/1.4 camera lenses, asingle f/2.8 micro lens such as a Nikon 60 mm Micro-Nikkor, or anycombination of lenses having a focal length ratio of 1:1.

The collection optics' magnification may be varied depending on theapplication. For example, the image resolution may be increased by afactor of 5 using a 5× collection optics. In one embodiment, the 5×collection optics may be a 5× microscope objective with 0.18 aperture,such as a model 80.3515 manufactured by Rolyn Optics, or any combinationof lenses 3250 and 3260 having a focal length ratio of 1:5.

A filter 3270, such as a 515 nm long pass filter, may be located betweenlenses 3250 and 3260 to block scattered laser light.

Collection optics 3255 image the fluorescence originating from thesurface of the substrate onto a CCD array 3300. In some embodiments, theCCD array may be a part of a CCD subsystem manufactured by Ocean OpticsInc. The subsystem, for example, may include a NEC linear CCD array andassociated control electronics. The CCD array comprises 1024 pixels(i.e., photodiodes), each of which is about 14 μm square (total activearea of about 14.4 mm×14 μm). Although a specific linear CCD array isdisclosed, it will be understood that any commercially available linearCCDs having various pixel sizes and several hundred to several thousandpixels, such as those manufactured by Kodak, EG&G Reticon, and Dalsa,may be used.

The CCD subsystem communicates with and is controlled by a dataacquisition board installed in computer 3400. Data acquisition board maybe of the type that is well known in the art such as a CIO-DAS16/Jrmanufactured by Computer Boards Inc. The data acquisition board and CCDsubsystem, for example, may operate in the following manner. The dataacquisition board controls the CCD integration period by sending a clocksignal to the CCD subsystem. In one embodiment, the CCD subsystem setsthe CCD integration period at 4096 clock periods. by changing the clockrate, the actual time in which the CCD integrates data can bemanipulated.

During an integration period, each photodiode accumulates a chargeproportional to the amount of light that reaches it. Upon termination ofthe integration period, the charges are transferred to the CCD's shiftregisters and a new integration period commences. The shift registersstore the charges as voltages which represent the light pattern incidenton the CCD array. The voltages are then transmitted at the clock rate tothe data acquisition board, where they are digitized and stored in thecomputer's memory. In this manner, a strip of the sample is imagedduring each integration period. Thereafter, a subsequent row isintegrated until the sample is completely scanned.

FIGS. 4a-4c illustrate flow cell 3220 in greater detail. FIG. 4a is afront view, FIG. 4b is a cross sectional view, and FIG. 4c is a backview of the cavity. Referring to FIG. 4a, flow cell 3220 includes acavity 3235 on a surface 4202 thereon. The depth of the cavity, forexample, may be between about 10 and 1500 μm, but other depths may beused. Typically, the surface area of the cavity is greater than the sizeof the probe sample, which may be about 13×13 mm. Inlet port 4220 andoutlet port 4230 communicate with the cavity. In some embodiments, theports may have a diameter of about 300 to 400 μm and are coupled to arefrigerated circulating bath via tubes 4221 and 4231, respectively, forcontrolling temperature in the cavity. The refrigerated bath circulateswater at a specified temperature into and through the cavity.

A plurality of slots 4208 may be formed around the cavity to thermallyisolate it from the rest of the flow cell body. Because the thermal massof the flow cell is reduced, the temperature within the cavity is moreefficiently and accurately controlled.

In some embodiments, a panel 4205 having a substantially flat surfacedivides the cavity into two subcavities. Panel 4205, for example, may bea light absorptive glass such as an RG1000 nm long pass filter. The highabsorbance of the RG1000 glass across the visible spectrum (surfaceemissivity of RG1000 is not detectable at any wavelengths below 700 nm)substantially suppresses any background luminescence that may be excitedby the incident wavelength. The polished flat surface of thelight-absorbing glass also reduces scattering of incident light,lessening the burden of filtering stray light at the incidentwavelength. The glass also provides a durable medium for subdividing thecavity since it is relatively immune to corrosion in the high saltenvironment common in DNA hybridization experiments or other chemicalreactions.

Panel 4205 may be mounted to the flow cell by a plurality of screws,clips, RTV silicone cement, or other adhesives. Referring to FIG. 4b,subcavity 4260, which contains inlet port 4220 and outlet port 4230, issealed by panel 4205. Accordingly, water from the refrigerated bath isisolated from cavity 3235. This design provides separate cavities forconducting chemical reaction and controlling temperature. Since thecavity for controlling temperature is directly below the reactioncavity, the temperature parameter of the reaction is controlled moreeffectively.

Substrate 130 is mated to surface 4202 and seals cavity 3235.Preferably, the probe array on the substrate is contained in cavity 3235when the substrate is mated to the flow cell. In some embodiments, anO-ring 4480 or other sealing material may be provided to improve matingbetween the substrate and flow cell. Optionally, edge 4206 of panel 4205is beveled to allow for the use of a larger seal cross section toimprove mating without increasing the volume of the cavity. In someinstances, it is desirable to maintain the cavity volume as small aspossible so as to control reaction parameters, such as temperature orconcentration of chemicals more accurately. In additional, waste may bereduced since smaller volume requires smaller amount of material toperform the experiment.

Referring back to FIG. 4a, a groove 4211 is optionally formed on surface4202. The groove, for example, may be about 2 mm deep and 2 mm wide. Inone embodiment, groove 4211 is covered by the substrate when it ismounted on surface 4202. The groove communicates with channel 4213 andvacuum fitting 4212 which is connected to a vacuum pump. The vacuum pumpcreates a vacuum in the groove that causes the substrate to adhere tosurface 4202. Optionally, one or more gaskets may be provided to improvethe sealing between the flow cell and substrate.

FIG. 4d illustrates an alternative technique for mating the substrate tothe flow cell. When mounted to the flow cell, a panel 4290 exerts aforce that is sufficient to immobilize substrate 130 locatedtherebetween. Panel 4290, for example, may be mounted by a plurality ofscrews 4291, clips, clamps, pins, or other mounting devices. In someembodiments, panel 4290 includes an opening 4295 for exposing the sampleto the incident light. Opening 4295 may optionally be covered with aglass or other substantially transparent or translucent materials.Alternatively, panel 4290 may be composed of a substantially transparentor translucent material.

In reference to FIG. 4a, panel 4205 includes ports 4270 and 4280 thatcommunicate with subcavity 3235. A tube 4271 is connected to port 4270and a tube 4281 is connected to port 4280. Tubes 4271 and 4281 areinserted through tubes 4221 and 4231, respectively, by connectors 4222.Connectors 4222, for example, may be T-connectors, each having a seal4225 located at opening 4223. Seal 4225 prevents the water from therefrigerated bath from leaking out through the connector. It will beunderstood that other configurations, such as providing additional portssimilar to ports 4220 and 4230, may be employed.

Tubes 4271 and 4281 allow selected fluids to be introduced into orcirculated through the cavity. In some embodiments, tubes 4271 and 4281may be connected to a pump for circulating fluids through the cavity. Inone embodiment, tubes 4271 and 4281 are connected to an agitation systemthat agitates and circulates fluids through the cavity.

Referring to FIG. 4c, a groove 4215 is optionally formed on the surface4203 of the flow cell. The dimensions of groove, for example, may beabout 2 mm deep and 2 mm wide. According to one embodiment, surface 4203is mated to the translation stage. Groove 4211 is covered by thetranslation stage when the flow cell is mated thereto. Groove 4215communicates with channel 4217 and vacuum fitting 4216 which isconnected to a vacuum pump. The pump creates a vacuum in groove 4215 andcauses the surface 4203 to adhere to the translation stage. Optionally,additional grooves may be formed to increase the mating force.Alternatively, the flow cell may be mounted on the translation stage byscrews, clips, pins, various types of adhesives, or other fasteningtechniques.

FIG. 5 illustrates an agitation system in detail. As shown, theagitation system 5000 includes two liquid containers 5010 and 5020,which in the some embodiments are about 10 milliliters each. Accordingto one embodiment, the containers may be centrifuge tubes. Container5010 communicates with port 4280 via tube 4281 and container 5020communicates with port 4270 via tube 4271. An inlet port 5012 and a ventport 5011 are located at or near the top of container 5010. Container5020 also includes an inlet port 5022 and a vent 5021 at or near itstop. Port 5012 of container 5010 and port 5022 of container 5020 areboth connected to a valve assembly 5051 via valves 5040 and 5041. Anagitator 5001, which may be a nitrogen gas (N₂) or other gas, isconnected to valve assembly 5051. Valves 5040 and 5041 regulate the flowof N₂ into their respective containers. In some embodiments, additionalcontainers (not shown) may be provided, similar to container 5010, forintroducing a buffer and/or other fluid into the cavity.

In operation, a fluid is placed into container 5010. The fluid, forexample, may contain targets that are to be hybridized with probes onthe chip. Container 5010 is sealed by closing port 5011 while container5020 is vented by opening port 5021. Next, N₂ is injected into container5010, forcing the fluid through tube 5050, cavity 2235, and finally intocontainer 5020. The bubbles formed by the N₂ agitate the fluid as itcirculates through the system. When the amount of fluid in container5010 nears empty, the system reverses the flow of the fluid by closingvalve 5040 and port 5021 and opening valve 5041 and port 5011. Thiscycle is repeated until the reaction between the probes and targets iscompleted.

The system described in FIG. 5 may be operated in an alternative manner.According to this technique, back pressure formed in the secondcontainer is used to reverse the flow of the solution. In operation, thefluid is placed in container 5010 and both ports 5011 and 5021 areclosed. As N₂ is injected into container 5010, the fluid is forcedthrough tube 5050, cavity 2235, and finally into container 5020. Becausethe vent port in container 5020 is closed, the pressure therein beginsto build as the volume of fluid and N₂ increases. When the amount offluid in container 5010 nears empty, the flow of N₂ into container 5010is terminated by closing valve 5040. Next, the circulatory system isvented by opening port 5011 of container 5010. As a result, the pressurein container 5020 forces the solution back through the system towardcontainer 5010. In one embodiment, the system is injected with N₂ forabout 3 seconds and vented for about 3 seconds. This cycle is repeateduntil hybridization between the probes and targets is completed.

b. Data acquisition

FIGS. 6-8 are flow charts describing one embodiment. FIG. 6 is anoverall description of the system's operation. A source code listingrepresentative of the software for operating the system is set forth inAppendix I which is included as a microfiche appendix.

At step 610, the system is initialized and prompts the user for testparameters such as:

a) pixel size;

b) scan speed;

e) scan temperature or temperatures;

c) number of scans to be performed;

d) time between scans;

f) thickness of substrate;

g) surface on which to focus;

h) whether or not to refocus after each scan; and

i) data file name.

The pixel size parameter dictates the size of the data points or pixelsthat compose the image. Generally, the pixel size is inversely relatedto the image resolution (i.e., the degree of discernable detail). Forexample, if a higher resolution is desired, the user would choose asmaller pixel size. On the other hand, if the higher resolution is notrequired, a larger pixel may be chosen. In one embodiment, the user maychoose a pixel size that is a multiple of the size of the pixels in theCCD array, which is 14 μm (i.e., 14, 28, 42, 56, etc.).

The scan-speed parameter sets the clock rate in the data acquisitionboard for controlling CCD array's integration period. The higher theclock speed, the shorter the integration time. Typically, the clock rateis set at 111 KHz. This results in an integration period of 36.9 ms(4096 clock periods per integration period).

The temperature parameter controls the temperature at which the scan isperformed. Temperature may vary depending on the materials being tested.The number-of-scans parameter corresponds to the number of times theuser wishes to scan the substrate. The time-between-scans parametercontrols the amount of time to wait before commencing a subsequent scan.These parameters may vary according to the application. For example, ina kinetics experiment (analyzing the sample as it approachesequilibrium), the user may configure the system to continuously scan thesample until it reaches equilibrium. Additionally, each scan may beperformed at a different temperature.

The thickness-of-substrate parameter, which is used by system'sauto-focus routine, is equal to the approximate thickness of thesubstrate that is being imaged. In some embodiments, the user optionallychooses the surface onto which the excitation light is to be focused(i.e., the front or back surface of the substrate). For example, thefront surface is chosen when the system does not employ a flow cell. Theuser may also choose to refocus the sample before beginning a subsequentscan. Other parameters may include specifying the name of the file inwhich the acquired data are stored.

At step 615, the system focuses the laser at the substrate. At step 620,the system initializes the x-y-z table at its start position. In someembodiments, this position corresponds to the edge of the sample atwhich the excitation line commences scanning. At step 625, the systembegins to translate the horizontal stage at a constant speed toward theopposite edge. At step 626, the CCD begins to integrate data. At step630, the system evaluates if the CCD integration period is completed,upon which the system digitizes and processes the data at step 640. Atstep 645, the system determines if data from all regions or lines havebeen collected. The system repeats the loop beginning at 626 until thesample has been completely scanned. At step 650, the system determinesif there are any more scans to perform, as determined by the set upparameters. If there are, the system calculates the amount of time towait before commencing the next scan at step 660. At step 665, thesystem evaluates whether to repeat the process from step 615 (ifrefocusing is desired) or 620 (if refocusing is not desired). Otherwise,the scan is terminated.

FIGS. 7a-7b illustrate focusing step 615 in greater detail.Auto-focusing is accomplished by the system in either two or threephases, depending upon which surface (front or back) the light is to befocused on. In the first phase, the system focuses the laser roughly onthe back surface of the substrate. At step 710, the system directs lightat one edge of the sample. The substrate reflects the light towardfocusing optics which directs it through vertical slit and at aphotodiode. As the substrate is translated through focus, the lightmoves horizontally across the vertical slit. In response to the light,the photo-diode generates a voltage proportional to the amount of lightdetected. Since the optics are aligned to locate the light in the middleof the slit when the substrate is in focus, the focus position willgenerally produce the maximum voltage.

At step 720, the system reads the voltage, and at step 725, compares itwith the previous voltage value read. If the voltage has not peaked(i.e., present value less then previous value), the system moves theflow cell closer toward the incident light at step 726. The distanceover which the flow cell is moved, for example, may be about 10 μm.Next, the loop beginning at step 720 is repeated until the voltagegenerated by the photodiode has peaked, at which time, the light isfocused roughly on the back surface. Because the flow cell is moved in10 μm steps, the focal plane of the light is slightly beyond the frontsurface (i.e., inside the substrate).

At step 728, the system determines at which surface to focus the light(defined by the user at step 610 of FIG. 6). If the front surface ischosen, the system proceeds to step 750 (the third focusing phase),which will be described later. If the back surface is chosen, the systemproceeds to step 730 (the second focusing phase).

At step 730, the system moves the flow cell closer toward the incidentlight. In some embodiments, the distance over which the flow cell ismoved is about equal to half the thickness of the substrate. Thisdistance is determined from the value entered by the user at step 610 ofFIG. 6. Generally, the distance is equal to about 350 mm, which is about1/2 the thickness of a typical substrate.

At step 735, the system reads the voltage generated by the photodiode,similarly as in step 720. At step 740, the system determines whether ornot the value has peaked. If it has not, the system moves the flow cellcloser toward the incident light at step 745. As in step 726, thedistance over which the flow cell is translated may be about 10 μm. Theloop commencing at step 735 is repeated until the voltage generated bythe photodiode has peaked, at which time, the laser is roughly focusedat a point beyond the front surface.

Next, the system starts the third or fine focusing phase, which focusesthe light at the desired surface. At step 750, the system moves the flowcell farther from the incident light, for example, in steps of about 1μm. The computer reads and stores the voltage generated by thephotodiode at step 755. At step 760, the encoder indicating the positionof the focus stage is read and the resulting data is stored. This valueidentifies the location of the focus stage to within about 1 μm. At step765, the system determines if the present voltage value is greater thenthe previous value, in which case, the loop at step 750 is repeated.According to some embodiments, the process beginning at 750 is repeated,for example, until the photodiode voltage is less than the peak voltageminus twice the typical peak-to-peak photodiode voltage noise. At step775, the data points are fitted into a parabola, where x=encoderposition and y=voltage corresponding to the position. At step 780, thesystem determines the focus position of the desired surface, whichcorresponds to the maximum of the parabola. By moving the flow cellbeyond the position at which the maximum voltage is generated andfitting the values to a parabola, effects of false or misleading valuescaused by the presence of noise are minimized. Therefore, this focusingtechnique generates greater accuracy than the method which merely takesthe position corresponding to the peak voltage.

At step 785, the system ascertains whether the opposite edge of thesubstrate has been focused, in which case the process proceeds to step790. Otherwise, the system moves the x-y-z translation stage in order todirect the light at the opposite edge at step 795. Thereafter, theprocess beginning at step 710 is repeated to focus second edge.

At step 790, the system determines the focus position of the othersubstrate position through linear interpolation using, for example, anequation having the following form: a+bx. Alternatively, a more complexmathematical model may be used to more closely approximate thesubstrate's surface, such as a+bx+cy+dxy.

By using the focusing method disclosed herein, the laser may be focusedon the front surface of the substrate, which is significantly lessreflective than the back surface. Generally, it is difficult to focus ona weakly reflective surface in the vicinity of a strongly reflectivesurface. However, this problem is solved by the present invention.

For embodiments employing a tilt stage, the focusing process can bemodified to focus the substrate vertically. The process is divided intotwo phases similar to the first and third focusing phase of FIGS. 7a-7b.

In the first phase, the tilt stage is initialized at an off-verticalposition and rotated, for example, in increments of about 0.1milliradians (mrad) toward vertical. After each increment, the voltagefrom photodiode (located behind the horizontal slit) is read and theprocess continues until the voltage has peaked. At this point, the tiltstage is slightly past vertical.

In the second phase, the tilt stage is rotated back toward vertical, forexample, in increments of about 0.01 mrad. After each rotation, thevoltage and corresponding tilt stage position are read and stored inmemory. These steps may be repeated until the voltage is less than thepeak voltage minus twice the typical peak-to-peak photodiode voltagenoise. Thereafter, the data are fitted to a parabola, wherein themaximum represents the position at which the substrate surface isvertical.

FIG. 8 illustrates the data acquisition process beginning at step 625 ingreater detail. In a specific embodiment, data are collected by scanningthe sample one vertical line at a time until the sample is completelyscanned. Alternatively, data may be acquired by other techniques such asscanning the substrate in horizontal lines.

At step 810, the x-y-z translation stage is initialized at its startingposition. At step 815, the system calculates the constant velocity ofthe horizontal stage, which is equal to the pixel size divided by theintegration period. Typically the velocity is about 0.3 mm/s.

At step 820, the system calculates the constant speed at which thefocusing stage is to be moved in order to maintain the substrate surfacein focus. This speed is derived from the data obtained during thefocusing phase. For example, the speed may be equal to:

    (F1-F2)/(P*N)

where F1=the focus position for the first edge; F2 is the focus positionof the second edge; P=the integration period; and N=the number of linesper scan.

At step 825, the system starts moving the translation stage in thehorizontal direction at a constant velocity (i.e., stage continues tomove until the entire two-dimensional image is acquired). At 826, thedata acquisition board sends clock pulses to the CCD subsystem,commencing the CCD integration period. At step 830, the systemdetermines if the CCD integration period is completed. After eachintegration period, the CCD subsystem generates an analog signal foreach pixel that is proportional to the amount of light sensed thereon.The CCD subsystem transmits the analog signals to the data acquisitionboard and begins a new integration period.

As the CCD subsystem integrates data for the next scan line, the dataacquisition board digitizes the analog signals and stores the data inmemory. Thereafter, the system processes the raw data. In someembodiments, data processing may include subtracting a line of darkdata, which represents the outputs of the CCD array in darkness, fromthe raw data. This compensates for the fact that the CCD output voltagesmay be non-zero even in total darkness and can be slightly different foreach pixel. The line of dark data may be acquired previously and storedin the computer's memory. Additionally, if the specified pixel size isgreater than 14 μm, the data are binned. For example, if the specifiedpixel size is 28 microns, the system bins the data 2 fold, i.e., the1024 data points are converted to 512 data points, each of whichrepresents the sum of the data from 2 adjacent pixels.

After the line of data is processed, it is displayed as a gray scaleimage. In one embodiment, the gray scale contains 16 gray levels.Alternatively, other gray scale levels or color scales may be used.Preferably, the middle of the scale corresponds to the average amount ofemission detected during the scan.

At step 830, the system determines if there are any more lines left toscan. The loop beginning at step 826 is repeated until the sample hasbeen completely scanned.

IV. Detailed Description of an Alternative Embodiment of the ImagingSystem

a. Detection Device

FIG. 9 schematically illustrates an alternative embodiment of an imagingsystem. As depicted, system 9000 comprises components which are commonto the system described in FIG. 3. The common components, for example,include the body 3220, fluid pump, vacuum pump, agitation system,temperature controller, and others which will become apparent. Suchcomponents will be given the same figure numbers and will not bediscussed in detail to avoid redundancy.

System 9000 includes a body 3220 on which a support 130 containing asample to be imaged is mounted. Depending on the application, the bodymay be a flow cell as described in FIGS. 4a-4c. The support may be matedto the body by vacuum pressure generated by a pump 3520 or by othermating technique. When attached to the body, the support and body sealsthe cavity except for an inlet port 3230 and an outlet port 3240. Fluidscontaining, for example, fluorescently labeled targets (fluorescein) areintroduced into cavity through inlet port 3230 to hybridize with thesample. A pump 3530 or any of the other fluid transfer techniquesdescribed herein may be employed to flow fluids into the cavity and outthrough outlet port 3240.

In some embodiments, an agitation system is employed to shorten theincubation period between the probes and targets by breaking up thesurface depletion layer above the sample. A temperature controller 3500may also be connected to the flow cell to enable imaging at the optimalthermal conditions. Computer 3400, which may be any appropriatelyprogrammed digital computer such as a Gateway 486DX operating at 33 MHz,operates the temperature controller.

Flow cell 3220 may be mounted on a three-axis (x-y-z) translation table3245. In some embodiments, the flow cell is mounted to the translationtable by vacuum pressure generated by pump 3250. To maintain the top andbottom of the probe sample in the focal plane of the incident light, theflow cell is mounted in a substantially vertical position. Thisorientation may be achieved by any of the methods described previously.

Movement of the translation table is controlled by a motion controller,which in some embodiments is a single axis motion controller fromPacific Precision Laboratories (PPL). In alternative embodiments, amulti-axis motion controller may be used to auto-focus the line of lighton the substrate or to enable other data collection schemes. The motioncontroller communicates and accepts commands from computer 3400.

In operation, light from an excitation source scans the substrate toobtain an image of the sample. Excitation source 9100 may be a model2065 argon laser manufactured by Spectra-Physics that generates about a3.0 mm diameter beam. The beam is directed through excitation opticsthat transform the beam to a line of about is about 15 mm×50 μm. Thisexcitation geometry enables simultaneous imaging of a row of the samplerather than on a point-by-point basis.

The excitation optics will now be described in detail. From the laser,the 3 mm excitation beam is directed through a microscope objective9120. For the sake of compactness, a mirror 9111, such as a 2" diameterNewport BD1, may be employed to reflect the incident beam to microscopeobjective 9120. Microscope objective 120, which has a magnificationpower of 10, expands the beam to about 30 mm. The beam then passesthrough a lens 9130. The lens, which may be a 150 mm achromat,collimates the beam.

Typically, the radial intensity of the expanded collimated beam has aGaussian profile. As previously discussed, scanning the support with anon-uniform beam is undesirable because the edges of the lineilluminated probe sample may appear dim. To minimize this problem, amask 9140 is inserted after lens 9130 for masking the beam top andbottom, thereby passing only the central portion of the beam. In oneembodiment, the mask passes a horizontal band that is about 7.5 mm.

Thereafter, the beam passes through a cylindrical lens 150 having ahorizontal cylinder axis, which may be a 100 mm f.l. made by MellesGriot. Cylindrical lens 9150 expands the beam spot vertically.Alternatively, a hyperbolic lens may be used to expand the beamvertically while resulting in a flattened radial intensity distribution.

From the cylindrical lens, the light passes through a lens 9170.Optionally, a planar mirror may be inserted after the cylindrical lensto reflect the excitation light toward lens 9170. To achieve the desiredbeam height of about 15 mm, the ratio of the focal lengths of thecylindrical lens and lens 9170 is approximately 1:2, thus magnifying thebeam to about 15 mm. Lens 9170, which in some embodiments is a 80 mmachromat, focuses the light to a line of about 15 mm×50 μm at thesample.

In a preferred embodiment, the excitation light irradiates the sample atan angle. This design decouples the illumination and collection lightpaths and improves the depth discrimination of the system.Alternatively, a confocal system may be provided by rotating theilluminating path about the collection optic axis to form a ring ofilluminating rays (ring illumination).

The excitation light causes the labeled targets to fluoresce. Thefluorescence is collected by collection optics. The collection opticsmay include lenses 9250 and 9260, which, for example, may be 200 mmachromats located nearly back to back at 1× magnification. Thisarrangement minimizes vignetting and allows the lenses to operate at theintended infinite conjugate ratio.

Collection optics direct the fluorescence through a monochromaticdepolarizer 9270, which in some embodiments is a model 28115manufactured by Oriel. Depolarizer 9270 eliminates the effect of thewavelength-dependent polarization bias of the diffraction grating on theobserved spectral intensities. Optionally, a filter 9280, which in someembodiments is a long-pass absorptive filter, may be placed afterdepolarizer 9270 to prevent any light at the incident wavelength frombeing detected. Alternatively, a holographic line rejection filter,dichroic mirror, or other filter may be employed.

The fluorescence then passes through the entrance slit of a spectrograph9290, which produces an emission spectrum. According to one embodiment,the spectrograph is a 0.5 Czerny-Turner fitted with toroidal mirrors toeliminate astigmatism and field curvature. Various diffraction gratings,such a 150/mm ruled grating, and 300/mm and 600/mm holographic gratingsare provided with the spectrograph.

The spectrograph's entrance slit is adjustable from 0 to 2 mm inincrements of 10 microns. By manipulating the width of the entranceslit, the depth of focus or axial response may be varied. FIG. 10illustrates the axial response of the line scanner as a function of slitwidth. As shown, a focus depth of about 50 microns is achieved with aslitwidth of 8 microns. In alternative embodiments, transmissiongratings or prisms are employed instead of a spectrograph to obtain aspectral image.

Referring back to FIG. 9, the spectrograph images the emission spectrumonto a spectrometric detector 9300, which may be a liquid cooled CCDarray detector manufactured by Princeton Instruments. Such CCD arraycomprises a 512×512 array of 25 μm pixels (active area of 12.8 mm×12.8mm) and utilizes a back-illuminated chip from Tektronix, thermostattedat -80° C. with 0.01° C. accuracy. Alternatively, a thermoelectricallycooled CCD or other light detector having a rectangular format may beused.

In some embodiments, CCD detector 9300 is coupled to and controlled by acontroller 9310 such as a ST 130 manufactured by Princeton Instruments.Controller 9310 interfaces with computer 3400 though a direct memoryaccess (DMA) card which may be manufactured by Princeton Instruments.

A commercially available software package, such as the CSMA softwarefrom Princeton Instruments, may be employed to perform data acquisitionfunctions. The CSMA software controls external devices via the serialand/or parallel ports of a computer or through parallel DATA OUT linesfrom controller 9310. The CSMA software enables control of various dataacquisition schemes to be performed, such as the speed in which an imageis acquired. The CCD detector integrates data when the shutter thereinis opened. Thus, by regulating the amount of time the shutter remainsopen, the user can manipulate the image acquisition speed.

The image's spatial and spectral resolution may also be specified by thedata acquisition software. Depending on the application, the binningformat of the CCD detector may be programmed accordingly. For example,maximum spectral and spatial resolution may be achieved by not binningthe CCD detector. This would provide spatial resolution of 25 micronsand spectral resolution of about 0.4 nm when using the lowest dispersion(150 lines/mm) diffraction grating in the spectrograph (full spectralbandpass of 80 nm at 150 lines/mm grating). Typically, the CCD is binned2-fold (256 channels) in the spatial direction and 8-fold (64 channels)in the spectral direction, which results in a spatial resolution of 50μm and spectral resolution of 3 nm.

If targets are labeled with fluorophores, continuous illumination ofsubstrate may cause unnecessary photobleaching. To minimizephotobleaching, a shutter 9110, which is controlled by a digital shuttercontroller 9420, is located between the light source and the directingoptics. Shutter 9110 operates in synchrony with the shutter inside theCCD housing. This may be achieved by using an inverter circuit 9421 toinvert the NOTSCAN signal from controller 9310 and coupling it tocontroller 9420. of course, a timing circuit may be employed to providesignals to effect synchronous operation of both shutters. In otherembodiments, photobleaching of the fluorophores may be avoided bypulsing the light source on in synchrony with the shutter in the CCDcamera.

Optionally, auto-focusing and/or maintaining the sample in the plane ofthe excitation light may be implemented in the same fashion as thesystem described in FIG. 3.

b. Data Acquisition

FIGS. 11a-11b are flow charts illustrating the steps for obtaining afull spectrally resolved image. A source code listing representative ofthe data acquisition software is set forth as follows:

Appendix II

    ______________________________________                                        SLITSCAN SOURCE LISTING                                                       ______________________________________                                        REM A program to acquire and rapidly write multiple                             REM frames of data to disk, moving PPL                                        REM stepper motor on 1 axis in between reads.                                 rem Receive and parse the number                                              rem of steps and stepper motor step size.                                     rem (1 um/step in closed loop, 0.2 um in open loop).                          int ctr                                                                       int numstep                                                                   long stepdist                                                                 let numstep = 256                                                             let stepdist = 50                                                             inp numstep, stepdist                                                         rem Convert microns to steps for open loop mode.                              let stepdist = 5*stepdist                                                     long left                                                                     int stepdig(8)                                                                let ctr = 8                                                                   let left = 0                                                                  while ctr > 0                                                                     let stepdig(ctr) = (stepdist-left)/power(10,ctr-1)                        let left = left + stepdig(ctr)*power(10,ctr-1)                                let ctr = ctr-1                                                             wend                                                                            REM **Initialize serial port & PPL motion controller**                        let tmp = SERIALINIT(1, 9600, 0, 8, 1)                                            let tmp = COMSEND(i,"e")                                                  let tmp = COMSEND(1,")                                                        let tmp = COMSEND(1,"m")                                                      let tmp = COMSEND(1,"n")                                                      let tmp = COMSEND(1,")                                                        let tmp = COMSEND(1,"j")                                                      let tmp = COMSEND(1,"0")                                                      let tmp = COMSEND(1,")                                                      rem Set up accel (turns/sec2) & vel. values (turns/sec)                         rem Any value from 1-50 and 1-10, respectively,                               rem is acceptable.                                                                let tmp = COMSEND(1,"a")                                                  let tmp = COMSEND(1,"1")                                                      let tmp = COMSEND(1,")                                                        let tmp = COMSEND(1,"v")                                                      let tmp = COMSEND(1,"1")                                                      let tmp = COMSEND(1,")                                                      rem Set up step distance.                                                           let tmp = COMSEND(1,"d")                                                  let tmp = COMSEND(1,stepdig(8)+48)                                            let tmp = COMSEND(1,stepdig(7)+48)                                            let tmp = COMSEND(1,stepdig(6)+48)                                            let tmp = COMSEND(1,stepdig(5)+48)                                            let tmp = COMSEND(1,stepdig(4)+48)                                            let tmp = COMSEND(1,stepdig(3)+48)                                            let tmp = COMSEND(1,stepdig(2)+48)                                            let tmp = COMSEND(1,stepdig(1)+48)                                            let tmp = COMSEND(1,")                                                      rem **INITIALIZE ARRAYS, etc**                                                  int a                                                                         let a = 0                                                                     int xprog                                                                     int yprog                                                                     int fastread                                                                  int proglines                                                                 int progstrips                                                                let rundone =                                                                 GETPROGXY (xprog, yprog, fastread, proglines, progstrips)                     unint image (yprog, xprog)                                                    float imflot (yprog,xprog)                                                    float bkflot (yprog, xprog)                                                   rem If we are in auto backgd subtraction mode, read                           rem it from disk.                                                             let backsup = GETPIV AR(97)                                                   if backsub = 1 then                                                               int backfile                                                              let tmp - GETASCII(25,backfile)                                               open backfile for input as 2                                                  unint bkg(yprog,xprog)                                                        read 2, bkg                                                                   if header(2,7) = 0 then                                                       let bkg = UNSCRAMBLE(bkg)                                                     endif                                                                       close 2                                                                         endif                                                                         rem **BEGIN DATA ACQUISITION**                                                int outfilenum                                                                let outfilenum = 1                                                            open EXPNAME for output as outfilenum                                         print ",0,0,0,11                                                              FOR framectr = 1 to numstep                                                       print "Collecting data",0,0,0,11                                          print framectr                                                                let tmp = INIT1FRAME                                                          let tmp = START130                                                            let a = WAIT1FRAME                                                            if a = 1 then                                                                           let tmp = STOP130                                                   iet tmp = GETFULLFRAME(image)                                                 if backsub = 1 then                                                                           let imflot = image                                            let bkflot = bkg                                                              let imflot = ABS(imflot - bkflot)                                             let image = imflot                                                          endif                                                                           let tmp = WRITEFRAME(outfilenum,image)                                            endif                                                                   rem Move stepper motor.                                                             let tmp = CCMSEND(1,"g")                                                  let tmp = COMSEND(1,")                                                      NEXT framectr                                                                   rem **WRITE HEADER INFO**                                                     rem Image written as unsigned integer data:                                   let a = SETHEADER(outfilenum,3,3)                                             Rem Store # of x points:                                                      let a = SETHEADER(outfilenum,4,xprog)                                         float totalstrips                                                             let totalstrips = (1.0*yprog) * (1.0*numstep)                                 let a = SETHEADER(outfilenum,5,totalstrips)                                   Rem Store # of y points:                                                      let a = SETHEADER(outfilenum,5,yprog)                                         Rem Image scrambled before storage:                                           let a = SETHEADER(outfilenum,7,1)                                             close outfilenum                                                              REM Return stepper to starting position                                       long dist                                                                     let dist = numstep*stepdjst + 1000                                            let ctr = 6                                                                   let left = 0                                                                  while ctr > 0                                                                     let stepdig(ctr) = (dist-left)/power(10,ctr-1)                            let left = left + stepdig(ctr)*power(10,ctr-1)                                let ctr = ctr-1                                                             wend                                                                            let tmp = COMSEND(1,"d")                                                      let tmp = COMSEND(1,"-")                                                      let tmp = COMSEND(1,stepdig(8)+48)                                            let tmp = COMSEND(1,stepdig(7)+48)                                            let tmp = COMSEND(1,stepdig(6)+48)                                            let tmp = COMSEND(1,stepdig(5)+48)                                            let tmp = COMSEND(1,stepdig(4)+48)                                            let tmp = COMSEND(1,stepdig(3)+48)                                            let tmp = COMSEND(1,stepdig(2)+48)                                            let tmp = COMSEND(1,stepdig(1)+48)                                            let tmp = COMSEND(1,")                                                        let tmp = COMSEND(1,"g")                                                      let tmp = COMSEND(1,")                                                        let tmp = COMSEND(1,"d")                                                      let tmp = COMSEND(1,"1")                                                      let tmp = COMSEND(1,"0")                                                      let tmp = COMSEND(1,"0")                                                      let tmp = COMSEND(1,"0")                                                      let tmp = COMSEND(1,")                                                        let tmp = COMSEND(1,"g")                                                      let tmp = COMSEND(1,")                                                        print ",0,0,0,11                                                              print "Data acquisition complete",0,0,0,11                                  ______________________________________                                    

At step 1110, the user configures the spectrograph for data acquisitionsuch as, but not limited to, defining the slit width of the entranceslit, the diffraction grating, and center wavelength of scan. Forexample the spectrograph may be configured with the followingparameters: 150/mm grating, 100 μm slitwidth, and between 570 to 600 nmcenter wavelength.

At step 1115, the user, through the CSMA data acquisition software andcontroller 310, formats the CCD detector. This includes:

a) number of x channels;

b) number of y channels;

c) CCD integration time; and

d) auto-background subtraction mode.

The number of x and y channels define the spectral and spatialresolution of the image respectively. Typically, the CCD is binned at256 channels in the spatial direction and 64 in the spectral direction.Using this configuration, the 12.8 mm×50 μm vertical strip from thesample is transformed into a series of 64 monochromatic images, eachrepresenting an 12.8 mm×50 μm image as if viewed through a narrowbandpass filter of about 3 nm at a specified center wavelength.

The CCD integration time parameter corresponds to the length of time theCCD acquires data. Typically, the integration period is set between 0.1to 1.0 seconds.

The auto-background subtraction mode parameter dictates whether abackground image is subtracted from the acquired data before they arestored in memory. If auto-background image is set, the system obtains abackground image by detecting the sample without illumination. Thebackground image is then written to a data file.

Subtracting the background image may be preferable because the CCDarrays used are inherently imperfect, i.e., each pixel in the CCD arraygenerally does not have identical operational characteristics. Forexample, dark current and ADC offset causes the CCD output to benon-zero even in total darkness. Moreover, such output may varysystematically from pixel to pixel. By subtracting the background image,these differences are minimized.

At step 1120, the user inputs parameters for controlling the translationstage, such as the number of steps and size of each step in which thetranslation stage is moved during the scanning process. For example, theuser defines the horizontal (or x) dimension of each pixel in the imagethrough the step size parameter. The pixel size in the x-direction isapproximately equal to the width of the sample divided by the number ofspatial channels in the y-direction. As an example, if the CCD is binnedat 256 spatial channels and the sample is about 12.8 mm×12.8 mm, then apixel size of 50 μm should be chosen (12.8 mm/256=50 μm).

At step 1125, the system initializes both the serial communication portof ST130 controller and PPL motion controller. At step 1130, the systemdefines an array in which the collected data are stored.

At step 1135, the system commences data acquisition by opening bothshutter and the CCD shutter. As the light illuminates the sample, thefluorophores emit fluorescence which is imaged onto the CCD detector.The CCD detector will generate a charge that is proportional to theamount of emission detected thereon. At step 1140, the system determinesif the CCD integration period is completed (defined at step 1115). TheCCD continues to collect emission at step 1135 until the integrationperiod is completed, at which time, both shutters are closed.

At step 1141, the system processes the raw data, Typically, thisinvolves amplification and digitization of the analog signals, which arestored charges. In some embodiments, analog signals are converted to 256intensity levels, with the middle intensity corresponding to the middleof analog voltages that have been detected.

At step 1145, the system determines if auto-background subtraction modehas been set (defined at step 1115). If auto background subtraction modeis chosen, the system retrieves the file containing the background imageat step 1150 and subtracts the background image from the raw data. atstep 1155. The resulting data are then written to memory at step 1160.On the other hand, if auto-background subtraction mode is not chosen,the system proceeds to step 1160 and stores the data in memory.

At step 1165, the system determines if there are any more lines of datato acquire. If so, the horizontal stage is translated in preparation forscanning the next line at step 1170. The distance over which thehorizontal stage is moved is equal to about one pixel width (defined atstep 1120). Thereafter, the system repeats the loop beginning 1135 untilthe entire area of the sample surface has been scanned.

c. Postprocessing of the Monochromatic Image Set

As mentioned above, the spectral line scanner is indispensable to thedevelopment of detection schemes such as those which simultaneouslyutilize multiple labels. A basic issue in any such scheme is how tohandle the spectral overlap between the labels. For example, if weexamine the fluorophores that are commercially available, we find thatany set that may be excited by the argon laser wavelengths will indeedhave substantial overlap of the emission spectra. The quantification ofthe surface coverages of these dyes clearly requires that imagesacquired at the various observation wavelengths be deconvoluted from oneanother.

The process of multi-fluorophore image deconvolution is formalized asfollows. The emission intensity I(λ_(i)) (photons cm⁻² s⁻¹ nm⁻¹)originating from a given region on the surface of the sample at anobservation wavelength λ_(i) is defined by:

    I(λ.sub.i)=I.sub.o Σσ.sub.ij ρ.sub.j

The variable I_(o) is the incident intensity (photons cm⁻² s⁻¹), ρ_(j)is the surface density (cm⁻²) of the j^(th) fluorophore species, andσ_(ij) is the differential emission cross section (cm² nm⁻¹) of thej^(th) fluorophore at the i^(th) detection wavelength. The system ofequations describing the observation of n fluorophore species at nobservation wavelengths may be expressed in matrix form as:

    I=I.sub.o σρ

Therefore, the surface density vector ρ (the set of surface densities inmolecules/cm² for the fluorescent species of interest) at each point onthe image can be determined by using the inverse of the emission crosssection matrix:

    ρ=(1/I.sub.o)σ.sup.-1 I

FIGS. 12a-12b are flow charts for deriving the relative cross sectionmatrix element. In particular, the process includes plotting thefluorescent emission spectrum from any region of the image that isobtained from the steps described in FIGS. 11a-11b. A source codelisting representative of the software for plotting the emission spectrais set forth in Appendix III which is included as a microfiche appendix.

At step 1210, the system prompts the user to input the name of the datafile of interest. The system then retrieves the specified data file. Thedata may be stored as a series of frames which, when combined, forms athree-dimensional image. As shown in FIG. 12c, each frame represents aspecific strip (12.8 mm×pixel width) of the sample at variouswavelengths. The x-axis corresponds to the spectrum; the y-axiscorresponds the vertical dimension of the sample; and the z-axisrepresents the horizontal dimension of the sample. By rearranging the xand z indices, so-called monochromatic images of the sample at specifiedobservation wavelengths are obtained. Further, the number of images isdetermined by the number of spectral channels at which the CCD isbinned. At step 1215, the system reads the data file and separates thedata into multiple 2-dimensional images, each representing an image ofthe sample at a specified observation wavelength.

At step 1220, the system displays an image of the sample at a specifiedobservation wavelength, each spatial location varying in intensityproportional to the fluorescent intensity sensed therein. At step 1225,the user selects a pixel or group of pixels from which a plot of theemission spectrum is desired. At step 1230, the system creates theemission spectrum of the selected pixel by extracting the intensityvalues of the selected regions from each image. Thereafter, the user mayeither plot the spectrum or sum the values of the present spectrum tothe previous spectrum at step 1235.

If the sum option is chosen, the system adds the spectra together atstep 1240 and proceeds to step 1245. On the other hand, if the plotoption is chosen, the system proceeds to step 1245 where the user maychoose to clear the plot (clear screen). Depending on the user's input,the system either clears the screen at step 1250 before plotting thespectrum at step 1255 or superimposes the spectrum onto an existing plotat step 1255. At step 1260, the system prompts the user to either endthe session or select another pixel to plot. If the user chooses anotherpixel to plot, the loop at step 1225 is repeated.

FIG. 13 illustrates the emission spectra of four fluorophores (FAM, JOE,TAMRA, and ROX) arbitrarily normalized to unit area. The scaling of thespectral intensities obtained from the procedure according to the stepsset forth in FIGS. 12a and 12b are proportional to the product of thefluorophore surface density and the excitation efficiency at the chosenexcitation wavelength (here either 488 nm or 514.5 nm). The excitationefficiencies per unit surface density or "unit brightness" of thefluorophores have been determined by arbitrarily scaling the emissionspectra to unit area. Consequently, the fluorophore densities obtainedtherefrom will reflect the arbitrary scaling.

For a four fluorophore system, the values of the emission spectra ofeach dye at four chosen observation wavelengths form a 4×4 emissioncross section matrix which can be inverted to form the matrix thatmultiplies four chosen monochromatic images to obtain four "fluorsurface density images".

FIG. 14 is a flow chart of the steps for spectrally deconvoluting thedata acquired during the data acquisition steps, as defined in FIGS.11a-11b. A source code listing representative of the software forspectral deconvolution is set forth in Appendix III.

Steps 1410 and 1420 are similar to 1210 and 1215 of FIGS. 12a-12b andtherefore will not be described in detail. At step 1410, the user inputsthe name of the data file from which the emission spectra is plotted. Atstep 1420, the system retrieves the data file and separates the datainto multiple images, each representing an image of the sample at aspecified observation wavelength.

At step 1430, the system queries the user for the name of the filecontaining the inverse emission cross section matrix elements. At step1440, the system retrieves the matrix file and multiplies thecorresponding inverse cross section matrix with the corresponding set offour monochromatic images. The resulting fluor density images are thenstored in memory at step 1450. At step 1460, the user may choose whichimage to view by entering the desired observation wavelength. At step1470, the system displays the image according to the value entered atstep 1460. At step 1480, the user may choose to view an image having adifferent observation wavelength. If another image is chosen, the loopbeginning at 1460 is repeated. In this manner, images depicting thesurface densities of any label may be obtained. This methodology enablesany spectral multiplexing scheme to be employed.

d. Example of spectral deconvolution of a 4-fluorophore system

FIG. 15 illustrates the layout of a VLSIPS array that was used todemonstrate spectral deconvolution. As shown, the array was subdividedinto four quadrants, each synthesized in a checkerboard pattern with acomplement to a commercial DNA sequencing primer. For example, quadrant1510 contains the complement to the T7 primer, quadrant 1520 containsthe complement to SP6 primer, quadrant 1530 contains the complement toT3 primer, and quadrant 1540 contains the complement to M13 primer.Further, each primer was uniquely labeled with a different fluorophorefrom Applied Biosystems (ABI). In this particular experiment, SP6 waslabeled with FAM (i.e. fluorescein), M13 was labeled with JOE (atetrachloro-fluorescein derivative), T3 was labeled with TAMRA (a.k.a.tetramethylrhodamine), and T7 was labeled with ROX (Rhodamine X). Inthis format, each quadrant of the VLSIPS array was labeled with just oneof the fluorophores. The array was then hybridized to a cocktail of thefour primers. Following a hybridization period in excess of 6 hours witha target concentration of 0.1 nanomolar, the array was washed with 6XSSPE buffer and affixed to the spectral line scanner flow cell.

The array was scanned twice with 80 mW of argon laser power at 488 nmand at 514.5 nm excitation wavelengths. The beam was focused to a line50 μm wide by 16 mm high. For each excitation wavelength, a series of256 frames was collected by the CCD and stored. The camera format was8-fold binning in the x or spectral direction and twofold binning in they direction, for a format of (x, y)=(64, 256). The x-axis motioncontroller was stepped 50 μm between frame acquisitions. Thespectrograph was configured with an entrance slitwidth of 100 μm s, a150 lines/mm ruled diffraction rating, and a central wavelength settingof 570 nm, producing a spectral bandpass from 480 nm to 660 nm.

The sets of spectral images were rearranged by interchanging the x andspectral indices to form two sets of 64 monochromatic images of thearray, each of which is characterized by a unique combination ofexcitation and observation wavelengths. The spectral bandwidth subsumedby each image is found by dividing the full spectral bandwidth by thenumber of images, i.e. (600 nm-480 nm)/64=2.8 nm. The monochromaticimages were rewritten as 8-bit intensities in the *.TIFF format.

FIG. 16 illustrates a set of four representative monochromatic spectralimages obtained from this experiment. Image 1601 was acquired with 488nm excitation light at an observation wavelength of 511 nm. This imagerepresents the emission generated by FAM. Image 1602, which was acquiredwith 488 nm excitation light at an observation wavelength of 553 nm,represents the signal emitted by JOE. Image 1603, which depicts the ROXsignal, was acquired with a 514.5 nm excitation light at an observationwavelength of 608 nm. As for Image 1604, it was acquired with 514.5 nmexcitation light at an observation wavelength of 578 nm. Image 1604represents the signal emitted by TAMRA.

The next step involves obtaining the emission spectrum of each dye.FIGS. 17-18 illustrate the spectra obtained from within each of the 4quadrants of the array at 488 and 514.5 excitation wavelengths,respectively. Since the labeled target molecules bound mutuallyexclusively to each quadrant of the array, each spectrum is a pureemission spectrum of just one of the ABI dyes. The observed differencesin the integrated areas of the raw spectra result from differences inexcitation efficiency and surface density of fluorophores. To increasethe value of the signals, the spectra may be normalized. FIG. 13illustrates the emission spectra of FIG. 17 after it has been normalizedto unit area.

Reliable spectral deconvolution may be achieved by choosing fourobservation wavelengths at or near the emission maxima of each of thefluorophores. Inspecting the images of FIG. 16, it can be seen that the510 nm image is sensitive only to the FAM dye. The other three are amixtures of the other three fluors. FIG. 19 is a spreadsheet showing thederivation of the relative inverse cross section matrix from the imagesof FIGS. 13.

FIG. 20 illustrates the "fluor surface density images", obtained bymultiplying the four chosen monochromatic images by the inverse of therelative emission cross section matrix. Images 2001, 2002, 2003, and2004 represent the relative surface density of JOE, ROX, TAMRA, and FAMrespectively. The signal and background levels in these images aresummarized on the Figure. As illustrated, a multi-labeled signal hasbeen deconvolved to provide signals, each substantially representing aunique label.

V. Detailed Description of Another Embodiment of the Imaging System

FIG. 21 illustrates an alternative embodiment of the present invention.The system shown in FIG. 21 employs air bearings to maintain the samplein the plane of the excitation light. System 2100 includes a body 1505on which a support 1500 containing a sample is mounted. In someembodiments, the body may be a flow cell that is of the type describedin FIG. 4a-4d. The body may be mounted to a single-axis translationtable so as to move the sample across the excitation light. Thetranslation table may be of the type already discloses in conjunctionwith the systems in FIGS. 3 and 9. Movement of the translation stage maybe controlled by a computer 1900.

An optics head assembly 2110 is located parallel to the sample. Theoptics head assembly may include components that are common with thosedescribed in FIG. 1. The common components are labeled with the samefigure numbers. To avoid being redundant, these components will not bediscussed here in detail. As shown, the optics head contains a lightsource 1100 for illuminating a sample 1500. Light source 1100 directslight through excitation optics 1200. The excitation optics transformthe beam to a line capable of exciting a row of the samplesimultaneously. In some applications, the light produced by theexcitation source may be nonhomogeneous, such as that generated by anarray of LEDs. In such cases, the excitation optics may employ lightshaping diffusers manufactured by Physical Optics Corporation, groundglass, or randomizing fiber bundles to homogenize the excitation light.As the light illuminates the sample, labeled markers located thereonfluoresce. The fluorescence are collected by collection optics 1300. Acollection slit 2131 may be located behind collection optics. In oneembodiment, the optics head is aligned such that substantially onlyemission originating from the focal plane of the light pass through theslit. The emission are then filtered by a collection filter 2135, whichblocks out unwanted emission such as illumination light scattered by thesubstrate. Typically, the filter transmits emission having a wavelengthat or greater than the fluorescence and blocks emission having shorterwavelengths (i.e., blocking emission at or near the excitationwavelength). The emission are then imaged onto an array of lightdetectors 1800. Subsequent image lines are acquired by translating thesample relative to the optics head.

The imaging system is sensitive to the alignment between the sample andplane of the excitation light. If the chip plane is not parallel to theexcitation line, image distortion and intensity variation may occur.

To achieve the desired orientation, the optics head is provided with asubstantially planar plate 2150. The plate includes a slit 2159,allowing the excitation and collection light paths to pass through. Anarray of holes 2156, which are interconnected by a channel 2155, arelocated on the surface 2151 of the plate. The channels are connected toa pump 2190 that blows air through the holes at a constant velocity. Theflow of air may be controlled via air flow valve 2195. The air creates apneumatic pressure between the support and the plate. By mounting theoptics head or the flow cell on a tilt stage, the pressure can beregulated to accurately maintain the plate parallel to the support. Insome embodiments, the air pressure may be monitored and controlled bycomputer 1900. A ballast 2196 may be provided in the air line to dampenany pressure variations.

In some embodiments, the head unit may be mounted on a single-axistranslation stage for focusing purposes. For example, the air pressuremay be monitored to accurately locate the sample in the focal plane ofthe excitation light. Alternatively, the imaging system may employ amulti-axis translation stage, focusing optics, and associated componentsfor focusing and scanning the sample, similar to the system disclosed inFIG. 3.

The present invention provides greatly improved methods and apparatusfor imaging a sample on a device. It is to be understood that the abovedescription is intended to be illustrative and not restrictive. Manyembodiments will be apparent to those of skill in the art upon reviewingthe above description.

Merely as an example, the focal lengths of the optical elements can bemanipulated to vary the dimensions of the excitation light or even tomake the system more compact. The optical elements may be interchangedwith other optical elements to achieve similar results such as replacingthe telescope with a microscope objective for expanding the excitationlight to the desired diameter. In addition, resolution of the image maybe manipulated by increasing or decreasing the magnification of thecollection optics.

The scope of the invention should, therefore, be determined not with thereference to the above description, but should instead be determinedwith reference to the appended claims, along with the full scope ofequivalents to which such claims are entitled.

What is claimed is:
 1. An apparatus for imaging a sample located on asupport, said apparatus comprising:a body for immobilizing said support,said support comprising at least a first surface having said samplethereon; an electromagnetic radiation source for generating excitationradiation having a first wavelength; excitation optics for transformingthe geometry of said excitation radiation to a line and directing saidline at said sample for exciting a plurality of regions thereon, saidline causing a labeled material on said sample to emit responseradiation, said response radiation having a second wavelength, saidfirst wavelength different from said second wavelength; collectionoptics for collecting said response radiation from said plurality ofregions; a detector for sensing said response radiation received by saidcollection optics, said detector generating a signal proportional to theamount of radiation sensed thereon, said signal representing an imageassociated with said plurality of regions from said sample; a translatorcoupled to said body for allowing a subsequent plurality of regions onsaid sample to be excited; a processor for processing and storing saidsignal so as to generate a 2-dimensional image of said sample; and afocuser for automatically focusing said sample in a focal plane of saidexcitation radiation.
 2. The apparatus as recited in claim 1 whereinsaid body comprises:a mounting surface; a cavity in said mountingsurface, said first surface mated to said mounting surface for sealingsaid cavity, said sample being in fluid communication with said cavity,said cavity having a bottom surface comprising a light absorptivematerial; an inlet and an outlet being in communication with said cavitysuch that fluid flowing into said cavity for contacting said sampleflows through said inlet and fluid flowing out of said cavity flowsthrough said outlet; and a temperature controller for controlling thetemperature in said cavity.
 3. The apparatus as recited in claim 2wherein said temperature controller comprises a thermoelectric cooler.4. The apparatus as recited in claim 1 wherein said excitation source isa laser.
 5. The apparatus as recited in claim 1 wherein said firstwavelength is selected to approximate the absorption maximum of the saidlabeled material used.
 6. The apparatus as recited in claim 1 whereinsaid excitation optics transform the geometry of said excitationradiation to a line having a length sufficient to excite a strip of saidsample with uniform intensity.
 7. The apparatus as recited in claim 6wherein said excitation optics comprises:a telescope for expanding andcollimating said excitation radiation; a cylindrical telescope forexpanding said excitation radiation from said telescope to a desiredheight; and a cylindrical lens for focusing said excitation radiationfrom said cylindrical telescope to a desired width at a focal plane ofsaid cylindrical lens.
 8. The apparatus as recited in claim 6 whereinsaid excitation optics comprises:a microscope objective for expandingsaid excitation radiation; a first lens for collimating said excitationradiation from said microscope objective, said lens comprising anachromatic lens; a cylindrical telescope for expanding said excitationradiation from said first lens to a desired height; and a second lensfor focusing said excitation radiation from said cylindrical telescopeto a desired width at a focal plane of said second lens, said secondlens comprising an achromatic lens.
 9. The apparatus as recited in claim1 further comprising a mirror for steering said excitation radiation toexcite said plurality of regions at a non-zero incident angle such thatsaid response radiation and said excitation line reflected from saidsupport are decoupled from each other.
 10. The apparatus as recited inclaim 9 wherein said non-zero incident angle is about 45 degrees. 11.The apparatus as recited in claim 1 wherein said collection optics havea magnification power sufficient to achieve a desired image resolution,said collection optics for imaging said response radiation onto saiddetector, said detector comprising a linear detector array having alength sufficient to detect said response emissions collected by saidcollection optics.
 12. The apparatus as recited in claim 11 wherein saidlinear detector comprises a CCD linear array.
 13. The apparatus asrecited in claim 1 wherein said translator comprises an x-y-ztranslation stage.
 14. The apparatus as recited in claim 1 wherein saidprocessor comprises a programmable digital computer.
 15. The apparatusas recited in claim 1 further comprising:a spectral detector forreceiving said response emission from said collection optics, saidspectral detector detecting a response emission spectrum; and a filterlocated in front of said spectral detector, said filter blockingradiation at said first wavelength and passing radiation at otherwavelengths.
 16. The apparatus as recited in claim 15 wherein saiddetector comprises a two-dimensional detector array having sufficientsize to detect said response emission spectrum from said plurality ofregions.
 17. The apparatus as recited in claim 16 wherein saidtwo-dimensional detector array comprises a two-dimensional CCD array.18. The apparatus as recited in claim 1 wherein said excitation sourceand said excitation optics are configured such that said line travelsalong a horizontal plane at said support.
 19. A method for imaging asample located on a support, said method comprising the stepsof:immobilizing said support on a body; exciting said sample on saidsupport with an excitation radiation having a first wavelength from anelectromagnetic radiation source, said excitation radiation having alinear geometry for exciting a plurality of regions on said sample;detecting a response radiation having a second wavelength in response tosaid excitation radiation, said response radiation representing an imageof said plurality of regions; exciting a subsequent plurality of regionson said sample; processing and storing said response radiation togenerate a 2-dimensional image of said sample; and auto-focusing saidsample in a focal plane of said excitation radiation.
 20. The method asrecited in claim 19 wherein said body comprises a mounting surfacehaving a cavity thereon, said support immobilized on said mountingsurface such that said sample is in fluid communication with saidreaction chamber, said reaction chamber comprising a inlet and a outletfor flowing fluids into and through said reaction chamber.
 21. Themethod as recited in claim 20 wherein said body further comprises atemperature controller for controlling the temperature in said cavity.22. The method as recited in claim 19 wherein said step of exciting saidsample comprises the step of directing said excitation radiation throughexcitation optics for transforming the excitation geometry of saidexcitation radiation to a line, said line having a length sufficient toexcite a strip of said sample with uniform energy and a width which isabout at least as narrow as the desired image resolution.
 23. The methodas recited in claim 17 wherein said step of detecting comprises thesteps of:collecting said response radiation through collection optics;and imaging said response radiation from the collection optics ontoradiation detectors, said radiation detectors comprising a linear CCDarray.
 24. The method as recited in claim 19 wherein said step ofexciting a subsequent plurality of regions comprises the step oftranslating said sample to allow said excitation radiation to excite asubsequent strip of said sample.
 25. The method as recited in claim 19wherein said step of processing and storing said response radiationcomprises the steps of:a) detecting said response radiation with adetector, said detector generating a signal proportional to amount ofradiation it senses; b) passing said signal to a processor, saidprocessor comprising a digital programmable computer; c) subtracting aline of dark data stored in said computer from said signal, said line ofdark data representing the signal generated by said detector when noradiation is present; d) storing said data from step c in a memory ofsaid computer; e) repeating steps a through d until the sample has beencompletely imaged; and e) combining the processed data to form a2-dimensional image of said sample.
 26. The method as recited in claim19 wherein said auto-focusing step comprises the steps of:a) focusing afirst surface of said support; b) focusing a second surface of saidsupport; and c) finely focusing said second surface.
 27. The method asrecited in claim 19 further comprising the step of detecting a responseradiation spectrum with a spectrometer, said spectrometer imaging saidspectrum onto a two-dimensional CCD array.